Glucagon derivatives

ABSTRACT

The present invention relates to a novel peptide of a glucagon derivative and a composition for preventing or treating obesity comprising the peptide as an active ingredient. The glucagon derivative according to the present invention shows a more excellent activating effect with regard to both glucagon-like peptide-1 receptors and glucagon receptors compared to native glucagon, and thus can be widely used as an effective agent for treating obesity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/684,110, filed Nov. 14, 2019, which is a divisional of U.S.application Ser. No. 15/539,238, filed Jun. 23, 2017 (U.S. Pat. No.10,513,550), which is the National Stage of International ApplicationNo. PCT/KR2015/014481, filed Dec. 30, 2015, which claims priority fromKorean Patent Application No. 10-2014-0193691, filed Dec. 30, 2014, thedisclosures of each of which are incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 12, 2021, isnamed 106132_000327_SL.txt and is 9 KB in size.

TECHNICAL FIELD

The present invention relates to a novel glucagon derivative having anexcellent effect on both glucagon-like peptide-1 (GLP-1) receptors andglucagon receptors, and a composition for preventing or treating obesitycontaining the glucagon derivative as an active ingredient.

BACKGROUND ART

Recent economic advances and lifestyle changes have been accompanied bygreat changes in dietary habit. In particular, busy people of today arebecoming overweight and obese due to high-calorie diets and insufficientexercise. According to a report of the World Health Organization (WHO),more than one billion adults are overweight worldwide, among them overthree million are clinically diagnosed with severe obesity, and 250,000people in Europe and 2.5 million people worldwide died of overweight- orobesity-related diseases every year (World Health Organization, GlobalStrategy on Diet, Physical Activity and Health, 2004).

Overweight and obesity increase blood pressure and blood cholesterollevel, thus becoming a cause of various diseases including heartdisease, diabetes, arthritis, etc., or aggravating the diseases.Further, overweight and obesity are some of the main causes thatincrease the risk of diseases such as arteriosclerosis, hypertension,hyperlipidemia, and heart disease in children and adolescents as well asin adults.

As such, obesity is now recognized as a serious disease prevalent allover the world and is a cause of various diseases. However, sinceobesity is believed to be overcome by self-help efforts, obesitypatients are being evaluated as people with low self-control.Nevertheless, obesity is not readily curable because it is a complicateddisease closely associated with appetite control and a mechanism ofaction for energy metabolism. Accordingly, for obesity treatment, it isrequired that both an individual effort for appetite control andtreatment of an abnormal mechanism of action for energy metabolism beconducted concurrently. In this regard, there has been a need for thedevelopment of a drug capable of treating the abnormal mechanism ofaction.

As a result of the above effort, anti-obesity drugs such as Rimonabant®(Sanofi-Aventis), Sibutramin® (Abbott), Contrave® (Takeda), Orlistat®(Roche), etc., have been developed. However, these drugs had drawbackssuch as fatal adverse reactions or little efficacy in treating obesity.For example, Rimonabant® shows an adverse reaction of central nervoussystem disorder, Sibutramin® and Contrave® show adverse cardiovasculareffects, and Orlist® shows an effect of body weight decrease of onlyabout 4 kg after one year of administration. Accordingly, there appearsto be no sure anti-obesity drug to be safely prescribed to obesitypatients.

As such, active research has been conducted to develop a newpharmaceutical drug to resolve the problems in the conventionalanti-obesity drugs, and recently, keen attention has been paid toglucagon derivatives. Glucagon is secreted by the pancreas when theblood glucose level falls low due to, for example, drug treatment,diseases, hormones, or enzyme deficiency. Glucagon signals the liver tobreak down glycogen to glucose and raise the blood glucose level toreturn to its normal level. Furthermore, glucagon has been reported tohave an anti-obesity effect, in addition to the effect of raising theblood glucose level, by suppressing appetite and activatinghormone-sensitive lipase in fat cells, thereby promoting fatdecomposition.

Glucagon-like peptide-1 (hereinafter, referred to as ‘GLP-1’), aglucagon derivative, is a substance under development as a drug toimprove hyperglycemia in diabetic patients. GLP-1 has the functions ofincreasing insulin synthesis and promoting its secretion, inhibitingglucagon secretion, inhibiting gastric emptying, enhancing the use ofglucose, and inhibiting food intake. Also, exendin-4, which is secretedby lizard venom and shows about 50% homology in amino acid sequence withGLP-1, is known to alleviate hyperglycemia in diabetes patients byactivating the GLP-1 receptor. However, anti-obesity drugs containingGLP-1 or exendin-4 have been reported to have adverse effects of causingvomiting and nausea.

In this regard, as a GLP-1 alternative, oxyntomodulin, which can bind toboth GLP-1 and glucagon peptides, has been highlighted. Oxyntomodulin isa peptide made from pre-glucagon, the precursor of glucagon, and has thesame effects of GLP-1 such as inhibiting food intake, promoting satiety,and fat decomposition, thus raising its potential as an anti-obesityagent.

However, oxyntomodulin or its derivatives have a drawback in that theyshould be administered daily at a high dose due to their short in vivohalf-lives and low efficacies.

DISCLOSURE OF INVENTION Technical Problem

The present inventors, in an effort to improve obesity treatment effectwhile reducing the dose, have developed a glucagon derivative with apartial modification in its amino acid sequence, and confirmed that theglucagon derivative has an excellent effect acting on both glucagonreceptors and GLP-1 receptors, thereby completing the present invention.

Solution to Problem

The present invention has been made keeping in mind the above problemsoccurring in the prior art, and an object of the present invention is toprovide a novel peptide showing an excellent obesity treatment effect.

Another object of the present invention is to provide a composition forpreventing or treating obesity containing the peptide.

Advantageous Effects of Invention

The novel peptide of the present invention can markedly activate bothGLP-receptors and glucagon receptors compared to the native glucagon,exhibiting an excellent anti-obesity effect even with a small amount ofadministration, and thus it can be widely used as a safe and effectiveagent for treating obesity.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to accomplish the above objects, in an aspect, the presentinvention provides a novel peptide having an amino acid sequence of thefollowing Formula 1:

(SEQ ID NO: 19) X1-X2-QGTFTSDYSKYL-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-W-L-X27-X28-X29 (Formula 1)

wherein X1 is histidine, desamino-histidyl, N-dimethyl-histidyl,β-hydroxy imidazopropionyl, 4-imidazoacetyl, β-carboxy imidazopropionyl,or tyrosine;

X2 is α-methyl-glutamic acid, aminoisobutyric acid (Aib), D-alanine,glycine, Sar(N-methylglycine), serine, or D-serine;

X15 is cysteine, aspartic acid, or glutamic acid;

X16 is glutamic acid, aspartic acid, serine, α-methyl-glutamic acid, orabsent;

X17 is cysteine, glutamine, glutamic acid, lysine, arginine, serine, orabsent;

X18 is cysteine, alanine, arginine, valine, or absent;

X19 is alanine, arginine, serine, valine, or absent;

X20 is lysine, histidine, glutamine, arginine, α-methyl-glutamic acid,or absent;

X21 is aspartic acid, glutamic acid, leucine, or absent;

X23 is isoleucine, valine, or absent;

X24 is arginine, alanine, cysteine, glutamic acid, lysine, glutamine,α-methyl-glutamic acid, or absent;

X27 is valine, alanine, lysine, methionine, glutamine, arginine, orabsent;

X28 is glutamine, lysine, asparagine, or absent; and

X29 is lysine, alanine, glycine, threonine, or absent;

with the proviso that the amino acid sequence identical to SEQ ID NO: 1is excluded.

The peptide of the present invention may include peptides, peptidederivatives, and peptide mimics thereof, which can activate both GLP-1receptors and glucagon receptors by modifying part of amino acid(s) viasubstitution.

As used herein, the term “native glucagon” refers to native humanglucagon having the amino acid sequence of

(SEQ ID NO: 1) His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln- Trp-Leu-Met-Asn-Thr.

The present invention provides the peptides defined above as derivativesof native glucagon, and in defining the peptides provided in the presentinvention, the peptides are intended to differ from native glucagon onlyat position(s) X in the sequences by alteration(s).

In the sequence of Formula 1 according to the present invention, aminoacids may be considered to be consecutively numbered from the firstamino acid to the 29^(th) amino acid in the conventional direction fromthe N-terminus to the C-terminus. Accordingly, the description on“position” in the sequence of Formula 1 should be interpreted in thesame manner as in the descriptions on positions of native human glucagonand other molecules.

As used herein, the term “peptide” refers to a compound in the formwhere two or more amino acids are linked by peptide bond(s). For thepurposes of the present invention, the peptide may refer to one whichexhibits an anti-obesity effect by activating both GLP-1 receptors andglucagon receptors.

Throughout the present invention, three-letter codes generally allowedfor different amino acids, such as α-aminoisobutyric acid (Aib),Sar(N-methylglycine), and α-methyl-glutamic acid, are used along withthe conventional one-letter or three-letter codes for naturallyoccurring amino acids.

Additionally, the amino acids described in the present invention areabbreviated as shown below according to IUPAC-IUB nomenclature.

alanine (A) arginine (R) asparagine (N) aspartic acid (D) cysteine (C)glutamic acid (E) glutamine (Q) glycine (G) histidine (H) isoleucine (I)leucine (L) lysine (K) methionine (M) phenylalanine (F) proline (P)serine (S) threonine (T) tryptophan (W) tyrosine (Y) valine (V)

The peptide, which has the amino acid sequence of Formula 1 according tothe present invention, may include any peptide that can activate bothglucagon receptors and GLP-1 receptors, via introduction ofsubstitution, addition, deletion, or modification after translation(e.g., methylation, acylation, ubiquitination, and intramolecularcovalent bonds) in the amino acid sequence of glucagon described by SEQID NO: 1.

For the substitution or addition of amino acids, atypical ornon-naturally occurring amino acids may be used, in addition to the 20amino acids conventionally observed in human proteins. Commercialproviders of the atypical amino acids include Sigma-Aldrich, ChemPep,Genzyme Pharmaceuticals, etc. The sequences for the peptides includingthese atypical amino acids and those for typical peptides may besynthesized or purchased from the commercial peptide manufacturingcompanies, e.g., American Peptide Company or Bachem (USA) or Anygen(Korea), etc.

For increasing the effect of the peptide of the present invention onglucagon receptors and GLP-1 receptors, in the amino acid sequencerepresented by SEQ ID NO: 1, the first amino acid, histidine, may besubstituted with 4-imidazoacetyl by deleting the α-carbon of histidine,substituted with desamino-histidyl by deleting the N-terminal aminegroup, substituted with N-dimethyl-histidyl by modifying the N-terminalamine group with two methyl groups, substituted with β-hydroxyimidazopropionyl by substituting the N-terminal amine group with ahydroxyl group, substituted with β-carboxy imidazopropionyl bysubstituting the N-terminal amine group with a carboxyl group, orsubstituted with tyrosine.

Additionally, the domain which binds to a GLP-1 receptor may besubstituted with an amino acid that can strengthen the hydrophobic bondand the ionic bond. Furthermore, a partial sequence of the glucagonsequence may be substituted with the amino acid sequence of GLP-1 or theamino sequence of exendin-4 to increase the activity of the GLP-1receptor.

Additionally, a partial sequence of the glucagon sequence may besubstituted with a sequence that can strengthen α-helix. Preferably, theamino acid(s) of the Formula 1 at positions 10, 14, 16, 20, 24, and 28may be substituted with the amino acid(s) composed of Tyr(4-Me), Phe,Phe(4-Me), Phe(4-Cl), Phe(4-CN), Phe(4-NO₂), Phe(4-NH₂), Phg, Pal, Nal,Ala(2-thienyl), or Ala(benzothienyl), which are known to assist inα-helix formation, or derivatives thereof. The kind and number of theamino acids or derivative thereof to be added for this purpose are notlimited.

Additionally, preferably, at least one amino acid in at least one aminoacid pair at positions 10 and 14, 12 and 16, 16 and 20, 20 and 24, and24 and 28 of the amino acid sequence of the Formula 1 may be substitutedwith glutamic acid or lysine, resulting in a pair of glutamic acid andlysine, which can form a ring, and the number of rings for insertion isalso not limited.

In an exemplary embodiment, the amino acid sequence of glucagon may besubstituted with a sequence having an ability to bind to GLP-1 receptorsso that the peptide can exhibit an excellent effect on both GLP-1receptors and glucagon receptors.

Preferably, the peptide of the present invention may be, in the aminoacid sequence of the Formula 1, a peptide,

wherein X1 is histidine;

X2 is α-methyl-glutamic acid;

X15 is cysteine or aspartic acid;

X16 is serine, glutamic acid, or aspartic acid;

X17 is arginine, lysine, glutamic acid, or cysteine;

X18 is cysteine, valine, or arginine;

X19 is alanine or valine;

X20 is glutamine, lysine, or histidine;

X21 is aspartic acid, glutamic acid, or leucine;

X23 is isoleucine or valine;

X24 is arginine, glutamic acid, or glutamine;

X27 is valine, lysine, or methionine;

X28 is glutamine, lysine, or asparagine; and

X29 is lysine, glycine, or threonine;

with the proviso that the amino acid sequence identical to SEQ ID NO: 1is excluded.

More preferably, the peptide of the present invention may be a peptideincluding an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOS: 2 to 14.

The peptide of the present invention may be prepared by a standardsynthesis method, a recombinant expression system, or any method knownin the art. Accordingly, the glucagon analogue according to the presentinvention may be synthesized by numerous methods including thefollowing:

(a) synthesizing a peptide by a step-wise method via a solid-phase orliquid-phase method, or a fragment assembly, separating the finalpeptide followed by purification;

(b) expressing a nucleic acid construct encoding the peptide in a hostcell, and recovering the expression product from the host cell culture;

(c) performing an expression of the peptide-encoding nucleic acidconstruct within a cell-free tube, and recovering the expressionproduct; or

(d) a method of obtaining fragments of a peptide by a random combinationof (a), (b), and (c), connecting the fragments, thereby recovering thecorresponding peptide.

The present inventors confirmed via in vitro experiments that thepeptide of the present invention has an excellent effect on GLP-1receptors and glucagon receptors, compared to native glucagon (see Table2). Additionally, via in vitro experiments it was confirmed that thepeptide of the present invention has an excellent inhibitory effectagainst feed intake in an obese animal model, thus demonstrating thatthe peptide of the present invention can exhibit an excellentanti-obesity effect even when a small amount is administered.

Accordingly, the peptide of the present invention is a dual agonistcapable of stimulating cAMP formation in both GLP-1 receptors andglucagon receptors, and is expected to have a more excellent effect oftreating obesity, compared to the existing glucagon. In this regard, thepeptide of the present invention can provide a more attractive selectionfor treating obesity and obesity-related diseases.

The peptide of the present invention, being a dual agonist, can combinethe effect of GLP-1 in food intake and the effect of glucagon in lipidmetabolism, and thereby synergistically act to accelerate the removal oflipid accumulation and continuous decrease of body weight. Thesynergistic effect as a dual agonist may help reduce cardiovascular riskfactors, such as high cholesterol and LDL, which may be completelyindependent of the effect on body weight.

Accordingly, the peptide of the present invention may be used as apharmaceutical drug for preventing weight increase, promoting weightdecrease, reducing overweight, and treating not only obesity includingmorbid obesity (e.g., via regulation of appetite, eating, food intake,calorie intake, and/or energy consumption) but also obesity-relateddiseases, including obesity-related inflammation, obesity-relatedgallbladder disease, obesity-induced sleep apnea, but not limitedthereto, and health conditions. Additionally, the peptide of the presentinvention may be used for the treatment of medical conditions that canbe associated with obesity, such as metabolic syndrome, hypertension,arteriosclerosis-inducing dyslipidemia, atherosclerosis,arteriosclerosis, coronary artery heart disease, stroke, etc. However,regarding these symptoms, the effect of the peptide of the presentinvention may be entirely or partially mediated through the bodyweight-related effects or may be independent of them.

In order to improve the therapeutic effect of the glucagon derivative ofthe present invention, the glucagon derivative may be modified using aconventional technique in the art, such as a modification of polymerssuch as polyethylene glycol (PEG), glycan, etc., or a fusion withalbumin, transferring, fatty acid, immunoglobulin, etc. For example, atleast one amino acid side chain in the compound of the present inventionmay be conjugated to a polymer in vivo so as to increase the solubilityand/or half-life and/or increase bioavailability. These modificationsare known to reduce the clearance of therapeutic proteins and peptides.

Preferably, the polymer may be water-soluble (amphipathic orhydrophilic), non-toxic, and pharmaceutically inactive, and morepreferably, may include PEG, a homopolymer or copolymer of PEG, amonomethyl-substituted polymer of PEG (mPEG), or a poly-amino acid suchas poly-lysine, poly-aspartic acid, and poly-glutamic acid.

It is obvious to those skilled in the art that the thus-modifiedglucagon derivatives have a more excellent therapeutic effect thannative glucagon. Accordingly, the variants of the glucagon derivativesare also included in the scope of the present invention.

In another aspect, the present invention provides a polynucleotideencoding the peptide.

As used herein, the term “homology”, used regarding polynucleotides,refers to a sequence similarity with a wild type amino acid sequence anda wild type nucleotide sequence, and includes gene sequences sharing atleast 75% with the polynucleotide sequence encoding the polypeptide,preferably at least 85%, more preferably at least 90%, and even morepreferably at least 95%. These homology comparisons may be performed bythe naked eye or using a comparison program which can be easilypurchased. The computer programs available on the market can calculatethe homologies between two or more sequences as a percentage. Thehomology (%) may be calculated on the neighboring sequences.

The peptide may be obtained in a large amount by inserting thepeptide-encoding polynucleotide into a vector followed by itsexpression.

In this kind of recombinant expression, the polynucleotide of thepresent invention is generally inserted into an appropriate vector, andforms a cloning or recombinant vector possessing the polynucleotide, andthe vector is also included in the scope of the present invention.

As used herein, the term “recombinant vector” refers to a DNA construct,which includes the nucleotide sequence of a polynucleotide encoding thetarget peptide which is operably linked to an appropriate regulatorysequence capable of expressing the target peptide in a suitable hostcell. The regulatory sequence may include a promoter capable ofinitiating transcription, an operator sequence for regulating thetranscription, a sequence encoding an appropriate mRNA ribosome-bindingdomain, and a sequence for regulating the termination of transcriptionand translation. The recombinant vector, once transformed into asuitable host cell, can replicate or function irrespective of the hostgenome, and may be integrated into the genome itself.

The recombinant vector to be used in the present invention is notparticularly limited, as long as it is replicable in a host cell, andmay be constructed using any vector known in the art. Examples of theconventional vectors to be used may include wild type or recombinantplasmids, cosmids, viruses, and bacteriophages. For example, as a phagevector or cosmid vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10,t11, Charon4A, Charon21A, etc., may be used. As a plasmid vector, apBR-based, a pUC-based, a pBluescriptII-based, a pGEM-based, apTZ-based, pCL-based, and a pET-based plasmid may be used. The vectorsto be used in the present invention are not particularly limited, butany vector known in the art may be used.

The recombinant vector may be used for the transformation of a host cellin order to produce the peptide of the present invention. Additionally,as part of the present invention, the transformed cell may be used forthe amplification of nucleic acid fragments or replication of vectors ofthe present invention, or a cultured cell or cell line used for therecombinant production of the peptide of the present invention.

As used herein, the term “transformation” refers to introduction of arecombinant vector including a polynucleotide encoding the targetprotein into a host cell so that the target protein encoded by thepolynucleotide can be expressed in the host cell. It does not matterwhether the polynucleotide is inserted to be positioned within thechromosome or outside the chromosome, as long as the transformedpolynucleotide can be expressed in the host cell.

Additionally, the polynucleotide includes DNA and RNA, which encode thetarget protein. The polynucleotide may be introduced in any form, aslong as the polynucleotide can be expressed after being introduced intoa host cell. For example, the polynucleotide may be introduced into ahost cell in the form of an expression cassette, which is a genomicstructure including all essential features required for self-expression.The expression cassette may generally include a promoter, which isoperably linked to the polynucleotide, a transcription terminationsignal, a ribosome-binding domain, and a translation termination signal.The expression cassette may be a self-replicable expression vector.Additionally, the polynucleotide itself may be inserted into a host celland operably linked to a sequence necessary for its expression in thehost cell, but is not limited thereto.

Additionally, as used herein, the term “operably linked” refers to astate, in which a promoter sequence, which initiates and mediatestranscription of the target protein-encoding polynucleotide, isfunctionally linked to the gene sequence.

The host cell suitable for the present invention is not particularlylimited, as long as the host cell can express the polynucleotide of thepresent invention. Examples of the host cells to be used in the presentinvention may include Escherichia sp. such as E. coli; Bacillus sp. suchas Bacillus subtilis; Pseudomonas sp. such as Pseudomonas putida; yeastssuch as Pichia pastoris, Saccharomyces cerevisiae, andSchizosaccharomyces pombe; insect cells such as Spodoptera frupperda(SF9); and animal cells such as CHO, COS, BSC, etc.

In another aspect, the present invention provides a pharmaceuticalcomposition for preventing or treating obesity containing the peptide asan active ingredient.

As used herein, the term “prevention” refers to any action resulting insuppression or delay of the onset of obesity by the administration ofthe peptide or the pharmaceutical composition of the present invention,and the term “treatment” refers to any action resulting in improvementin symptoms of obesity or the beneficial alteration by theadministration of the peptide or the pharmaceutical composition of thepresent invention.

As used herein, the term “administration” refers to introducing aparticular substance to a patient in an appropriate manner. Theadministration route of the pharmaceutical composition of the presentinvention, although not particularly limited, may be any of the commonroutes, as long as the pharmaceutical composition can reach the targettissue in the body, e.g., intraperitoneally, intravenously,intramuscularly, subcutaneously, intradermally, orally, topically,intranasally, intrapulmonarily, intrarectally, etc.

As used herein, the term “obesity” refers to a medical condition inwhich excess body fat has accumulated in the body, and people areconsidered as obese when the body mass index (BMI; a measurementobtained by dividing a person's weight in kilograms divided by thesquare of height in meters) is 25 or higher. Obesity is generallyinduced by energy imbalance due to calorie intake being higher thanenergy consumption. Obesity is a metabolic disease, which can inducediabetes and hyperlipidemia, increase the risk of sexual dysfunction,arthritis, and cardiovascular disease, and in some cases, is alsoassociated with the occurrence of cancer.

The pharmaceutical composition of the present invention may include apharmaceutically acceptable carrier, excipient, or diluent.

As used herein, the term “pharmaceutically acceptable” refers to asufficient amount, which can exhibit a therapeutic effect but does notincur any adverse reactions, and may be easily determined by thoseskilled in the art according to factors known in the medicinal field,such as the type of diseases to be treated, the patient's age, weight,gender, sensitivity to drugs, administration routes, number ofadministration, drug(s) to be combined or concurrently used, etc.

The pharmaceutical composition of the present invention may furtherinclude a pharmaceutically acceptable carrier. For oral administration,the pharmaceutically acceptable carrier may include, although is notlimited to, a binder, a lubricant, a disintegrator, an excipient, asolubilizer, a dispersing agent, a stabilizer, a suspending agent, acoloring agent, and a perfume. For injectable administration, abuffering agent, a preserving agent, an analgesic, a solubilizer, anisotonic agent, and a stabilizer may be mixed for use. For topicaladministration, the pharmaceutically acceptable carrier may include abase, an excipient, a lubricant, a preserving agent, etc.

The pharmaceutical composition of the present invention may beformulated into various dosage forms in combination with thepharmaceutically acceptable carrier. For example, for oraladministration, the pharmaceutical composition may be formulated intotablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.For injectable administration, the pharmaceutical composition may beformulated into an ampoule as a unit dosage form or a multi-doseadministration. The pharmaceutical composition may be also formulatedinto solutions, suspensions, tablets, pills, capsules, and long-actingpreparations.

On the other hand, examples of the carrier, excipient, and diluentsuitable for the pharmaceutical composition of the present invention mayinclude lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calciumphosphate, calcium silicate, cellulose, methylcellulose,microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate,mineral oils, etc. In addition, the pharmaceutical composition of thepresent invention may further include fillers, anti-coagulating agents,lubricants, humectants, perfumes, antiseptics, etc.

Additionally, the pharmaceutical composition may be formulated into oneselected from the group consisting of tablets, pills, powders, granules,capsules, suspensions, liquid medicine for internal use, emulsions,syrups, sterile aqueous solutions, non-aqueous solvents, freeze-driedformulations, and suppositories.

Additionally, the pharmaceutical composition may be formulated into asuitable formulation for unit administration into a patient's bodyaccording to the conventional method, preferably into a formulation typeuseful for the administration of a peptide drug, and administered orallyor parenterally via a subcutaneous, intravenous, intramuscular,intraarterial, intermedullary, intrathecal, intraventricular,intrapulmonary, intradermal, subcutaneous, intraperitoneal, intranasal,intragastric, local, sublingual, intravaginal, or intrarectal routeaccording to the conventional method, but is not limited thereto.

Additionally, the peptide may be used by mixing with various carriers,such as a saline solution or an organic solvent, which are accepted aspharmaceutical drugs. For increasing stability or absorbency, thepeptide may be used along with carbohydrates such as glucose, sucrose ordextran, or antioxidants such as glutathione, chelating agents, lowmolecular weight proteins, or other stabilizers, etc.

The amount and number of administration of the pharmaceuticalcomposition of the present invention may be determined according to thetypes of drugs as active ingredients, along with other factors such asthe diseases to be treated, administration routes, the patient's age,gender, weight, severity of the illness, etc.

The total effective dose of the composition of the present invention maybe administered to a patient as a single dose, or as a multiple dose fora long-term period according to the fractionated treatment protocol. Thepharmaceutical composition of the present invention may have a differentcontent of the active ingredient according to the severity of thedisease. Preferably, the total dose of the peptide of the presentinvention may be about 0.0001 μg to 500 mg per 1 kg of the patient'sbody weight. However, regarding the dose of the peptide, the effectivedose is determined considering various factors such as the patient'sage, weight, health conditions, gender, severity of illness, diet andexcretion rate, etc., those skilled in the art can determine theappropriate effective dose according to the particular use of thecomposition of the present invention.

The formulations, administration routes, and administration methods ofthe pharmaceutical composition of the present invention may not beparticularly limited, as long as the pharmaceutical composition can showthe effect of the present invention.

Since the pharmaceutical composition of the present invention has anexcellent in vivo duration and titer, the number and frequency ofadministration of the pharmaceutical composition of the presentinvention may be significantly reduced.

The pharmaceutical composition may be administered alone or incombination with other pharmaceutical formulations exhibiting an effectof preventing or treating obesity. The pharmaceutical formulationexhibiting an effect of preventing or treating obesity may include,although is not particularly limited to, a GLP-1 receptor agonist, aleptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, amelanin-concentrating hormone (MCH) receptor antagonist, a Y2/3 receptoragonist, an MC3/4 receptor agonist, a gastric/pancreatic lipaseinhibitor, a 5HT2c agonist, a 3A receptor agonist, an amylin receptoragonist, a ghrelin antagonist, and/or a ghrelin receptor antagonist,etc.

In another aspect, the present invention provides a method forpreventing or treating obesity including administering the peptide or apharmaceutical composition containing the peptide to a subject.

As used herein, the term “subject” refers to a subject suspected ofhaving obesity or being at risk for having obesity, and specifically,referring to mammals, including humans, rats, and cattle, but thesubject may be any subject that can be treated by the peptide of thepresent invention, without limitation. The administration of apharmaceutical composition containing the peptide of the presentinvention can effectively treat a subject suspected of having obesity,and the obesity is the same as described above.

The therapeutic method of the present invention may includeadministering a pharmaceutically effective amount of the pharmaceuticalcomposition containing the peptide. The total daily dose of thecomposition can be determined through appropriate medical judgment by aphysician, and the composition may be administered once or in a fewdivided doses. However, in view of the purpose of the present invention,the specific therapeutically effective dose of the composition for anyparticular patient may vary depending on various factors well known inthe medical field, including the kind and degree of responses to beachieved, specific compositions according to whether or not other agentsare used therewith, the patient's age, body weight, health conditions,gender and diet, time and route of administration, the discharge rate ofthe composition, the duration of treatment, other drugs used incombination or concurrently with the composition of the presentinvention, and other factors known in the medical field.

In still another aspect, the present invention provides a use of thepeptide, in preparing a pharmaceutical drug for preventing or treatingobesity.

MODE FOR THE INVENTION

Hereinafter, the present invention will be described in more detail withreference to the following Examples. However, these Examples are forillustrative purposes only, and the invention is not intended to belimited by these Examples.

EXAMPLE 1: PRODUCTION OF CELL LINES FOR IN VIVO ACTIVATION <1-1>Production of Cell Lines Showing cAMP Response to GLP-1

A PCR reaction was performed using the open reading frame (ORF) of cDNA(OriGene Technologies, Inc., USA) of a human GLP-1 receptor gene as atemplate along with the forward and reverse primers represented by SEQID NOS: 15 and 16, which include the restriction sites for HindIII andEcoRI, respectively.

In particular, the PCR reaction was performed (denaturing at 95° C. for60 seconds, annealing at 55° C. for 60 seconds, and elongation at 68° C.for 30 seconds) for 30 cycles. The PCR product was electrophoresed on a1.0% agarose gel, and a 405 bp fragment was obtained therefrom viaelution.

forward primer: (SEQ ID NO: 15) 5′-CCCGGCCCCCGCGGCCGCTATTCGAAATAC-3′reverse primer: (SEQ ID NO: 16) 5′-GAACGGTCCGGAGGACGTCGACTCTTAAGATAG-3′

The PCR product was cloned into a known animal cell expression vector,x0GC/dhfr (Korea Patent No. 10-0880509, the same herein after), toconstruct a recombinant vector, x0GC/GLPIR.

The thus-constructed recombinant vector, x0GC/GLPIR, was transformedinto cells of the Chinese hamster ovary cell line CHO DG44, which werecultured in a DMEM/F12 medium containing 10% FBS, using Lipofectamine(Invitrogene, USA), and selected and cultured in a selective mediumcontaining G418 (1 mg/mL) and methotraxate (10 nM). Monoclone cell lineswere selected therefrom, and among them, the cell lines showingexcellent cAMP responses to GLP-1 in a dose-dependent manner werefinally selected.

<1-2> Production of Cell Lines Showing cAMP Response to Glucagon

A PCR reaction was performed using the open reading frame (ORF) of cDNA(OriGene Technologies, Inc., USA) of a human glucagon receptor gene as atemplate along with the forward and reverse primers represented by SEQID NOS: 17 and 18, which include the restriction sites for EcoRI andXhoI, respectively.

In particular, the PCR reaction was performed (denaturing at 95° C. for60 seconds, annealing at 55° C. for 60 seconds, and elongation at 68° C.for 30 seconds) for 30 cycles. The PCR product was electrophoresed on a1.0% agarose gel, and a 435 bp fragment was obtained therefrom viaelution.

forward primer: (SEQ ID NO: 17) 5′-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3′reverse primer: (SEQ ID NO: 18) 5′-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3′

The PCR product was cloned into the known animal cell expression vector,x0GC/dhfr, to construct a recombinant vector, x0GC/GCCR.

The thus-constructed recombinant vector, x0GC/GCCR, was transformed intocells of the Chinese hamster ovary cell line CHO DG44, which werecultured in a DMEM/F12 medium containing 10% FBS, using Lipofectamine(Invitrogene, USA), and selected and cultured in a selective mediumcontaining G418 (1 mg/mL) and methotraxate (10 nM). Monoclone cell lineswere selected therefrom, and among them, the cell lines showingexcellent cAMP responses to glucagon in a dose-dependent manner werefinally selected.

EXAMPLE 2: SYNTHESIS OF GLUCAGON DERIVATIVES

In order to develop a glucagon derivative having an excellent effect onboth GLP-1 receptors and glucagon receptors, the amino acid sequence ofnative glucagon represented by SEQ ID NO: 1 was substituted with anamino acid sequence which has an ability to bind to GLP-1 receptors, andglucagon derivatives were synthesized as shown in Table 1 below.

TABLE 1 SEQ ID NO Amino Acid Sequence SEQ ID NO: 1HSQGTFTSDYSKYLDSRRAQDFVQWLMNT SEQ ID NO: 2 HXQGTFTSDYSKYLDEKCAKEFIQWLVNTSEQ ID NO: 3 HXQGTFTSDYSKYLDEKCVKLFIQWLVNT SEQ ID NO: 4HXQGTFTSDYSKYLDEKCAKEFVEWLVNT SEQ ID NO: 5 HXQGTFTSDYSKYLDEKCAHEFVEWLVNTSEQ ID NO: 6 HXQGTFTSDYSKYLDSKCAHEFVEWLVNT SEQ ID NO: 7HXQGTFTSDYSKYLDSKCVHEFIEWLKNT SEQ ID NO: 8 HXQGTFTSDYSKYLDSKCAHEFIEWLKNKSEQ ID NO: 9 HXQGTFTSDYSKYLDSECAHEFIEWLKQG SEQ ID NO: 10HXQGTFTSDYSKYLDDKCAHEFVEWLVNT SEQ ID NO: 11HXQGTFTSDYSKYLDEECAKEFIRWLKKG SEQ ID NO: 12HXQGTFTSDYSKYLCEKRAKEFVQWLMNT SEQ ID NO: 13HXQGTFTSDYSKYLDECRAKEFVQWLMNT SEQ ID NO: 14HXQGTFTSDYSKYLDEKCAKEFVQWLMNT

In Table 1 above, the amino acid indicated as “X” in the sequences ofSEQ ID NOS: 2 to 14 represents α-methyl-glutamic acid, which is anon-native amino acid, and the lysine residue in these sequences canform a ring with glutamic acid residue.

EXAMPLE 3: MEASUREMENT OF IN VITRO ACTIVITY OF GLUCAGON DERIVATIVES

In order to measure the anti-obesity activities of the glucagonderivatives synthesized in Example 2, the in vitro cellular activitiesof the glucagon derivatives were measured using the transformed celllines prepared in Examples 1-1 and 1-2.

The transformed cell lines were prepared so that the human GLP-1receptor gene and the human glucagon receptor gene can be expressed inCHO, respectively, and are suitable for measuring the activities ofGLP-1 and glucagon. Accordingly, the activities of glucagon derivativessynthesized according to the present invention were measured using thetransformed cell lines, respectively.

Specifically, the transformed cell lines were subcultured two or threetimes per each week, aliquoted into a 96-well plate with 1×10⁵cells/well, and cultured for 24 hours, respectively.

The cultured cells were washed with Krebs-Ringer Bicarbonate (KRB)buffer solution, suspended in 40 mL of KRB buffer solution containing 1mM 3-isobutyl-1-methylxanthine (IBMX), and placed at room temperaturefor 5 minutes.

The native glucagon (SEQ ID NO: 1) or glucagon derivatives(representatively, peptides of SEQ ID NOS: 12 to 14) according to thepresent invention were subjected to serial dilution at 5-fold intervalsranging from 1000 nM to 0.02 nM, 40 mL of the above cells were addedthereto, and cultured in a CO₂ incubator at 37° C. for 1 hour.

Then, 20 mL of cell lysis buffer was added to the respective resultants,and the cell lysates were applied to a cAMP assay kit (Molecular Device,USA) to measure cAMP concentration, and EC₅₀ values were calculated andcompared therebetween. The results are shown in Table 2 below.

TABLE 2 EC₅₀ (nM) Test Material hGLP-IR hGCGR GLP-1 0.36 >1,000glucagon >1,000 1.48 SEQ ID NO: 12 0.96 1.38 SEQ ID NO: 13 0.27 0.23 SEQID NO: 14 0.17 0.38

As shown in Table 2 above, the glucagon derivatives according to thepresent invention showed excellent effects on both GLP-1 receptors andglucagon receptors, compared to the native glucagon represented by SEQID NO: 1.

Glucagon is known to have an obesity treatment effect by activatingGLP-1 receptors and glucagon receptors, thereby suppressing appetite,improving satiety, and promoting fat cell lysis. Since the glucagonderivatives according to the present invention are now shown to haveexcellent in vitro effects on both GLP-1 receptors and glucagonreceptors, compared to the native glucagon, these glucagon derivativescan be used as a more effective agent for treating obesity than theexisting glucagon.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

What is claimed:
 1. A peptide comprising the amino acid sequence of SEQID NO:
 14. 2. The peptide of claim 1, wherein the peptide is capable ofactivating a GLP-1 receptor and a glucagon receptor.
 3. The peptide ofclaim 1, wherein the peptide has an anti-obesity effect.
 4. The peptideof claim 1, wherein at least one amino acid pair at positions 12 and 16or 16 and 20 of the peptide forms a ring.
 5. A pharmaceuticalcomposition comprising the peptide of claim 1 as an active ingredient.6. The pharmaceutical composition of claim 5, further comprising apharmaceutically acceptable carrier.
 7. The pharmaceutical compositionof claim 5, wherein the pharmaceutical composition is administered aloneor in combination with other pharmaceutical formulations exhibiting aneffect of treating obesity.
 8. The pharmaceutical composition of claim7, wherein the other pharmaceutical formulation exhibiting an effect oftreating obesity is a GLP-1 receptor agonist, a leptin receptor agonist,a DPP-IV inhibitor, a Y5 receptor antagonist, a melanin-concentratinghormone (MCH) receptor antagonist, a Y2/3 receptor agonist, an MC3/4receptor agonist, a gastric/pancreatic lipase inhibitor, a 5HT2cagonist, a 3A receptor agonist, an amylin receptor agonist, a ghrelinantagonist, or a ghrelin receptor antagonist.
 9. A method for treatingobesity, comprising administering the peptide of claim 1 to a subject inneed thereof.
 10. A method for treating obesity, comprisingadministering the pharmaceutical composition of claim 5 to a subject inneed thereof.